Porous polymeric matrices made of natural polymers and synthetic polymers and optionally at least one cation and methods of making

ABSTRACT

A porous polymeric matrix containing at least one natural polymer and at least one synthetic polymer and optionally at least one cation. Furthermore, a method of making a porous polymeric matrix involving mixing at least one natural polymer and inorganic salts with a solution comprising at least one solvent and at least one synthetic polymer to form a slurry, casting the slurry in a mold and removing the solvent to form solid matrices, immersing the solid matrices in deionized water to allow natural polymer cross-linking and pore creation to occur simultaneously, and drying the matrices to create a porous polymeric matrix; wherein the matrix contains a cation. Also, a method of making a porous polymeric matrix, involving mixing at least one natural polymer in an aqueous solvent and mixing at least one synthetic polymer in an organic solvent, combining the mixtures and casting in a mold, and separately removing said aqueous solvent and said organic solvent to form a porous polymeric matrix; wherein the porous polymeric matrix does not contain a cation.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/578,618, filed 10 Jun. 2004, and U.S. Provisional Application No.60/613,936, filed 28 Sep. 2004, which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates to a porous polymeric matrix containing atleast one natural polymer and at least one synthetic polymer andoptionally at least one cation. Furthermore, the present inventionrelates to a method of making a porous polymeric matrix involving mixingat least one natural polymer and inorganic salts with a solutioncontaining at least one solvent and at least one synthetic polymer toform a slurry, casting the slurry in a mold and removing the solvent toform solid matrices, immersing the solid matrices in deionized water toallow natural polymer cross-linking and pore creation to occursimultaneously, and drying the matrices to create a porous polymericmatrix; wherein the matrix contains a cation. Also, a method of making aporous polymeric matrix, involving mixing at least one natural polymerin an aqueous solvent and mixing at least one synthetic polymer in anorganic solvent, combining the mixtures and casting in a mold, andseparately removing said aqueous solvent and said organic solvent toform a porous polymeric matrix; wherein the porous polymeric matrix doesnot contain a cation.

Polymers such as poly(lactide-co-glycolide)(p(LGA)) has been usedclinically for tissue repair and organ regeneration for decades.Poly(lactide-co-glycolide), a hydrophobic polymer, is biocompatible,biodegradable, and easily processed into a variety of sizes and shapeswhich have good mechanical properties (Ma, P. X., and R. Langer,Fabrication of biodegradable polymer foams for cell transplantation andtissue engineering, In: Tissue engineering methods and protocols, J.Morgan and M. Yarmush, editors, Humana Press Inc., Totowa, N.J., 1999,p. 47-56; Lanza, R. P., et al., Principles of tissue engineering,Academic Press, San Diego, Calif., 1997; Patrick, C. W., et al.,editors, Frontiers in tissue engineering, Pergamon, N.Y., 1998; Ma, P.X., et al., J. Biomed. Mater. Res., 54(2): 284-93 (2001)). Althoughp(LGA) will support cell attachment and cell growth, it does not impartsignals to the cells (Langer, R., J. P. Vacanti, Science, 260: 920-6(1993)). The inability to carry signal molecules limits the applicationof polymers such as p(LGA). This deficiency is currently overcome bysynthesizing block or graft copolymers of lactic acid and lysine orother segments carrying side chain functional groups (Langer, R., J. P.Vacanti, Science, 260: 920-6 (1993); Ouchi, T. et al, Macromolecules,3(5):885-8 (2002)). Through the functional groups, specific amino acidsequences can be attached. By this strategy, a number of new chemicalentities have been provided. However, the preparation of such copolymersinvolves a series of cumbersome isolation, purification andidentification procedures.

The present invention provides matrices, made of natural polymers (e.g.,pectins) and synthetic polymers such as p(LGA) and optionally at leastone cation, that retain the biomechanical strength of polymers such asp(LGA) yet also provide access for hydrophilic, bioactive substances.

SUMMARY OF THE INVENTION

In accordance with the present invention there is provided a porouspolymeric matrix containing at least one natural polymer and at leastone synthetic polymer and optionally at least one cation. Furthermore,in accordance with the present invention there is provided a method ofmaking a porous polymeric matrix involving mixing at least one naturalpolymer and inorganic salts with a solution containing at least onesolvent and at least one synthetic polymer to form a slurry, casting theslurry in a mold and removing the solvent to form solid matrices,immersing the solid matrices in deionized water to allow natural polymercross-linking and pore creation to occur simultaneously, and drying thematrices to create a porous polymeric matrix; wherein the matrixcontains a cation. Also, a method of making a porous polymeric matrix,involving mixing at least one natural polymer in an aqueous solvent andmixing at least one synthetic polymer in an organic solvent, combiningthe mixtures and casting in a mold, and separately removing said aqueoussolvent and said organic solvent to form a porous polymeric matrix;wherein the porous polymeric matrix does not contain a cation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SEM photographs of (A) pectin particles and (B) NaCl—CaCl₂mixtures (bar=100 μm).

FIG. 2 shows (A) SEM images of p(LGA) matrix showing a continuousnetwork of salt cavities with the size of 50-200 μm (bar=100 μm), (B)Pectin/p(LGA) composite matrix showing the leaf- or sheet-like pectinstructures which stretched over all space (bar=100

FIG. 3 shows (A) averaged confocal reflection images instereo-projection of pectin/p(LGA) indicating pectin domains constructedwith irregular flat sheets with mid-line ridges or small flat patches(1), p(LGA) domains of fine network of anastomosing fibers (2), and theareas of both (3), (B) Laser confocal micrograph of fluorescentlylabeled pectin/p(LGA) showing the fluorescence located in pectin areas,not in p(LGA) areas (bar=100 mm).

FIG. 4 shows typical plots of (A) storage modulus, (B) loss modulus, and(C) loss tangent as a function of temperature; the pectin/p(LGA)composite matrix (∘) has higher storage modulus and loss modulus valuesthan p(LGA) matrix (•) and pectin matrix (▾) in the −80° C. to −40° C.range; the p(LGA) matrix has a glass transition at about 50° C., abovethat the p(LGA) gives no force reading, in contrast some residual forcestill remained with pectin/p(LGA) matrix and pectin matrix; the losstangent cures of all three types of matrices show a similar trend.

FIG. 5 shows time curves of water adsorption (A) and protein adsorption(B) in pectin/p(LGA) matrix (∘) and p(LGA) matrix (•), the experimentswere conducted at room temperature using PBS as an incubation media, theprotein concentration in PBS was 0.1%, w/v.

FIG. 6 shows comparison of pectin/p(LGA) to p(LGA) matrices inwater/protein adsorption; A₁ and A₂, the amount of adsorbates detectedat equilibrium in pectin/p(LGA) and p(LGA) matrices, respectively.

FIG. 7 shows osteoblast distribution in pectin/p(LGA) matrices (toppanel) and p(LGA) matrices (bottom panel) after 1 day cell seeding (thesamples were stained using hematoxylin and eosin), there were moreosteoblasts (arrow head) in pectin/p(LGA) matrices than in p(LGA)matrices (magnification: A (40×) and B (200×)).

FIG. 8 shows in vitro osteoblast proliferation on (▴) pectin/p(LGA) and(▪) p(LGA) matrices versus cultivation time, one million cells wereseeded onto each matrix, P<0:01.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a porous polymeric matrix containing atleast one natural polymer and at least one synthetic polymer andoptionally at least one cation. Furthermore, the present inventionrelates to a method of making a porous polymeric matrix involving mixingat least one natural polymer and inorganic salts with a solutioncomprising at least one solvent and at least one synthetic polymer toform a slurry, casting the slurry in a mold and removing the solvent toform solid matrices, immersing the solid matrices in deionized water toallow natural polymer cross-linking and pore creation to occursimultaneously, and drying the matrices to create a porous polymericmatrix; wherein the matrix contains a cation. Also, a method of making aporous polymeric matrix, involving mixing at least one natural polymerin an aqueous solvent and mixing at least one synthetic polymer in anorganic solvent, combining the mixtures and casting in a mold, andseparately removing said aqueous solvent and said organic solvent toform a porous polymeric matrix; wherein the porous polymeric matrix doesnot contain a cation.

The present invention provides three-dimensional porous matrices(scaffolds) made of natural polymers such as pectin and water insolublesynthetic polymers such as p(LGA) and optionally at least one cation.The matrices retain the biomechanical strength of the syntheticpolymers, such as p(LGA), yet provides access for hydrophilic, bioactivesubstances.

The synthetic polymers which may be utilized in the present inventionmay be non-biodegradable or biodegradable synthetic water insolublepolymers. The biodegradable polymers may be polyesters, polyanhydrides,or polyortho esters. Representative polyesters include poly(lacticacid), poly(glycolic acid), and poly(lactic acid-co-glycolic acid),poly(lactide) [p(LA)], poly(glycolide) [p(GA)],poly(lactide-co-glycolide) [p(LGA)], polycaprolactone andpoly(lactide-co-caprolactone). The preferred polyester is p(LGA).Representative polyanhydrides include poly(carboxyphenoxypropane-sebacic acid) [p(CPP/SA), CPP:SA=20:80 and 50:50],poly[1,6-bis(p-carboxyphenoxy)hexane [p(CPH)], andpoly(anhydride-co-imide), the preferred polyanhydride is p(CPP/SA).Representative polyortho esters include the polyortho esters prepared bycondensation polymerization of3,9-diethylidene-2,4,8,10-tetraoxaspiroundecane (DETOSU) and the diolscontaining trans-cyclohexanedimethanol, triethylene glycol orN-methyldiethanolamine, and their copolymers with p(LGA) or polyethyleneglycol; the preferred polyortho esters are their copolymer containingp(LGA). Other biodegradable polymers include polyamides, such aspolypeptides; poly(butyric acid), poly(valeric acid), and theircopolymers, polyalkylene glycols such as poly(ethylene glycol),polyalkylene oxides such as poly(ethylene oxide). Non-biodegradablepolymers include polyalkylene terepthalates such as poly(ethyleneterephthalate), polyvinyl alcohols, polyvinyl halides such as poly(vinylchloride), polyvinylpyrrolidone, polysiloxanes, poly(vinyl acetate),polystyrene, polyurethanes, polycarbonates, polyalkylenes such aspolyethylene and polypropylene, and co-polymers thereof. Polymers ofacrylic acid, methacrylic acid or copolymers or derivatives thereofincluding, poly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate) jointly referred to herein as “polyacrylic acids”), and blendsthereof. As used herein, “derivatives” include polymers havingsubstitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art.

Preferably the synthetic polymer is poly(lactide-co-glycolide) with anyMW or MW polydispersity, all ratios between lactic acid (LA) andglycolic acid (GA), and all degrees of crystallinity. Generally, the MWranges from about 500 to about 10,000,000 Da, preferably from about2,000 to about 1,000,000 Da, and more preferably from about 500 to about5,000 Da. The p(LGA) with the ratio of LA:GA at about 75:25 to about85:15 (mol:mol) and the MW from about 5,000 to about 500,000 arepreferred for matrices designed for all tissue repair, especially forbone repair.

Representative natural polymers and their derivatives include thefollowing: proteins such as albumin and polysaccharides such as pectin,chitosan, hyaluronate esters, polyhydroxybutyrate, cellulose andderivativized celluloses such as alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulphate sodium salt (jointly referred to herein as “syntheticcelluloses”). Preferably the natural polymer is pectin; the molecularweight (MW) range of pectin used depends on the degree of pectinesterification (DE; a designation of the percent of carboxyl groupsesterified with methanol). Generally the MW of the pectin ranges fromabout 500 to about 1,000,000 Da (preferably from about 230,000 to about280,000 Da; more preferably about 3000 Da); MW of pectin was measured byhigh performance size exclusive chromatography (HPSEC) equipped withon-line multi-angle laser light scattering and viscometric detection(Fishman, M. L., et al., J. Agr. Food Chem., 49: 4494-4501 (2001)).Generally the DE ranges from about 10 to about 100% (preferably fromabout 25 to about 76%; more preferably from about 25 to about 35%).

A solvent for the polymer is selected based on its biocompatibility aswell as the solubility of the polymer and where appropriate, interactionwith the agent to be delivered. For example, the ease with which theagent is dissolved in the solvent and the lack of detrimental effects ofthe solvent on the agent to be delivered are factors to consider inselecting the solvent. Aqueous solvents can be used to make matricesformed of water-soluble polymers. Organic solvents will typically beused to dissolve hydrophobic and some hydrophilic polymers. Preferredorganic solvents are volatile or have a relatively low boiling point orcan be removed under vacuum and which are acceptable for administrationto humans in trace amounts, such as methylene chloride. Other solvents,such as ethyl acetate, ethanol, methanol, dimethyl formamide (DMF),acetone, acetonitrile, tetrahydrofuran (THF), acetic acid, dimethylsulfoxide (DMSO) and chloroform, and combinations thereof, also may beutilized. Preferred solvents are those rated as class 3 residualsolvents by the Food and Drug Administration, as published in theFederal Register vol. 62, number 85, pp. 24301-24309 (May 1997).Dichloromethane is preferably used with polylactide-co-glycolide.

In general, the polymer is dissolved in the solvent to form a polymersolution having a concentration of between 0.1 and 60% weight to volume(w/v), more preferably between 0.25 and 30%. The polymer solution isthen processed as described below to yield a polymer matrix.

Generally, the matrices are produced by a novel method involving thefollowing:

(a) Dispersing at least one natural polymer (e.g., pectin) and inorganicsalt particles into a solution containing at least one synthetic polymerand at least one solvent (e.g., p(LGA)/dichloromethane) and mixing wellto form a slurry; generally at a temperature of from about 4 to about37° C. (preferably from about 10 to about 27° C.; more preferably atabout 20° C.) and for about 1 to about 30 minutes (preferably from about3 to about 20 minutes; more preferably from about 3 to about 5 minutes).The ratio of the salt particles and natural polymer (e.g., pectin) tothe synthetic polymer (e.g., p(LGA)) ranges from about 1:1 to about 40:1(preferably about 20:1, more preferably about 9:1 (w/w). The inorganicsalts used are dependant on the type of natural polymer utilized;generally the inorganic salt may be calcium chloride, sodium chloride,magnesium chloride, sodium sulfate, potassium sulfate, ammonium sulfate,ammonium chloride, potassium chloride, or others known in the art. Forexample, with pectin the salts may be calcium chloride or a mixture ofcalcium chloride and sodium chloride or a mixture of magnesium chlorideand sodium chloride; preferably calcium chloride and/or sodium chloride.Sodium nitrate, potassium nitrate, or magnesium nitrate many be usedwith calcium chloride when the natural polymer is pectin or alginate orhyaluronate or carboxy methyl cellulose. The ratio of natural polymer(e.g., pectin) to salt generally ranges from about 1:about 0.1-about 20(preferably about 1:15, more preferably about 1:about 10). The size ofnatural polymers (e.g., pectin) and salt particles are generally about20 to about 300 microns (preferably about 30 to about 250 microns; morepreferably about 50 to about 200 microns).

(b) Casting the slurry in a mold and removing the solvent by evaporation(or by other methods known in the art) to form solid matrices (generallyat a temperature of from about 4 to about 37° C., preferably from about10 to about 27° C., preferably at about 20° C.; generally bench+vacuumranges from (12 to 24)+(2 to 8), more preferably (18 to 20)+(2 to 4),more preferably 18+4).

(c) Immersing the solid matrices in a large volume of deionized (DI)water thus allowing pectin cross-linking and pore creation to occursimultaneously (generally at room temperature). The amount of DI wateris generally from about 0.5 to about 50 L (preferably about 0.5 to about40 L, more preferably about 0.5 L) and the volume of matrix is generallyfrom about 1 to about 10 ml (preferably about 2 to about 8 ml, morepreferably about 5 ml).

(d) Drying (e.g., freeze drying or by other methods known in the art)the matrices to create a channeled porous structure. Generally freezedrying is utilized at a temperature from about −5 to about −70° C.(preferably about −10 to about −50° C., more preferably about −20° C.).

The ratio of natural polymer (e.g., pectin) to synthetic polymer isflexible and generally ranges from about 0.1:about 99.9 to about99.9:about 0.1 (pectin:polymer); about 5-about 15 parts of pectin toabout 100 parts of polymer is preferred.

Suitable applications for the present matrices (scaffolds) will varywith polymer composition and structure. For example, biodegradablepolymer scaffolds are suitable for use in either in vitro applicationsand/or in vivo cell transplantation. The matrices may serve then assupports or scaffolds to allow cell growth to occur in vitro prior toimplantation in vivo. The scaffolds may also be used directly in vivo,without being pre-seeded with cells. In both applications (with orwithout prior cell seeding), biodegradable polymer matrices inaccordance with the present invention are particularly useful for thegrowth of three-dimensional tissue and may be used in the growth ofconnective tissues, like bone, cartilage, paradontal tissue, as well asdental tissues and other organs, such as liver or breast tissue.

For example, any desired cell type may be cultured in vitro in thepresence of one of the matrices of the present invention to form amatrix that is coated, impregnated or infiltrated with the cells.Preferably, the cells are derived from a mammal, and most preferablyfrom a human. In one example, fibroblast infiltrated matrices may beplaced at the site of a skin lesion (e.g., wound or ulcer) to promotehealing of the lesion. Other cell types that can be cultured on thematrices of this invention include but are not limited to, osteocytes,chondrocytes, keratinocytes, and tenocytes. Matrices impregnated withthese cells can be used to aid in the healing of bone, cartilage, skin,and tendons and ligaments, respectively. Matrices can also be generatedwhich contain a mixture of cell types, e.g., to mimic the cellularmakeup of a desired tissue. The matrices of this invention can also beseeded with non-differentiated mesenchymal cells that can differentiateinto a variety of tissue specific types upon implantation, or seededwith fetal or neonatal cells of the desired type. One advantageassociated with the use of the cellular matrices in vivo is that thematrix is completely biocompatible and is reabsorbed by the body.Alternatively, matrices impregnated with various cell types are usefulfor in vitro diagnostic applications. For example, matrices infiltratedwith fibroblasts can be used to test the efficacy and/or toxicity ofvarious pharmaceutical or cosmetic compounds.

In one aspect, the invention features a method for promoting cell growthand proliferation in vitro. In this aspect, the method includes thesteps of obtaining a sample of cells, admixing the cells with thematrices described herein, and then culturing the admixture underconditions sufficient to promote growth and infiltration of the cellsinto the matrix. Cells which may be grown according to the method of theinvention include any cell type which can be cultured in vitro;preferably, the cells are mammalian; and most preferably, they arederived from a human.

In still another aspect, the invention includes a method for promotingcell growth and proliferation in vivo at the site of an injury, e.g., ina mammal, preferably a human. This method includes the steps ofobtaining a sample of cells capable of promoting healing of the injury,admixing the cells with the matrices described herein, and placing theadmixture at the site of injury in the mammal to promote growth andproliferation of cells at the site in order to facilitate the healing ofthe injury.

Embodiments of this aspect of the invention include obtaining the cellsample directly from the desired tissue and admixing the sample with thematrices described herein; obtaining the cell sample from the desiredtissue and culturing the cells in vitro prior to admixture with thematrices described herein; and obtaining the cell sample from anestablished cell line and admixing the cells with the matrices describedherein. Preferably, the admixture containing the cell sample and thematrix is cultured in vitro under conditions sufficient to promoteproliferation and infiltration of the cells into the matrix prior toplacement at the site of injury.

The cells admixed with the matrix for this aspect of the invention canbe of any cell type which is capable of supporting cell growth andproliferation at the site of injury. For example, the source of thecells can be xenogeneic to the mammal, but preferably the cells areallogeneic, and most preferably the cells are immunologically compatiblewith the mammal. Further, the infiltrated matrix can contain cells ofthe same cell type as the cells found at the site of injury (e.g., fromthe same tissue), or the matrix can contain cells which are of adifferent cell type but which deposit extracellular matrix componentswithin the matrix to serve as a scaffold for cell growth in vivo.

In preferred embodiments of this aspect of the invention, the cells arefibroblasts and the infiltrated matrix is placed at the site of a skinlesion (e.g., a wound, burn, surgical incision, or a dermal ulcer), thecells are osteocytes, and the infiltrated matrix is placed at the siteof a bone injury; the cells are chondrocytes and the infiltrated matrixis placed at the site of an injury to cartilaginous tissue; the cellsare keritinocytes and the infiltrated matrix is placed at the site of askin lesion; the cells are tenocytes and the infiltrated matrix isplaced at the site of an injury to a tendon; or the cells arenon-differentiation mesenchymal cells.

The matrices of the invention may further include a drug for use as adrug delivery system. The particular drug used is a matter of choicedepending on the intended use of the composition. Preferred drugsinclude, but are not limited to, proteins (e.g., growth factors,enzymes), steroids, non-steroidal anti-inflammatory drugs, cytotoxicagents (e.g., anti-tumor drugs), antibiotics, oligonucleotides (e.g.,antisense), and biopolymers. When provided for cell and tissue growthand proliferation, the matrices of the invention may further includegrowth factors, and cell attachment proteins or peptides.

The matrix used in the methods of the invention can further contain oneor more drugs, e.g., a growth factor to further enhance growth of thecells and/or an antibiotic to reduce the risk of infection at the siteof placement. Active agents which can be incorporated into the matrixfor delivery include therapeutic or prophylactic agents. These can beproteins or peptides, sugars, oligosaccharides, nucleic acid molecules,or other synthetic or natural agents. The agents may be labeled with adetectable label such as a fluorescent label or an enzymatic orchromatographically detectable agent. Preferred drugs includeantibiotics, antivirals, vaccines, vasodilators, vasoconstrictors,immunomodulatory compounds, including steroids, antihistamines, andcytokines such as interleukins, colony stimulating factors, tumornecrosis factor and interferon (alpha, beta, gamma), oligonucleotidesincluding genes and antisense, nucleases, bronchodilators, hormonesincluding reproductive hormones, calcitonin, insulin, erythropoietin,growth hormones, and other types of drugs such as Antiban™.

In another aspect, the invention features a porous polymeric matrixcontaining at least one natural polymer (e.g., described above) and atleast one synthetic polymer (e.g., described above) without any organicor inorganic cross-linker or any divalent metal ions (e.g., calciumchloride). One method to make such a porous polymeric matrix involvesmixing at least one natural polymer in an aqueous solution and at leastone synthetic polymer in an organic solvent (e.g., described above) toform an emulsified system, casting the emulsified polymers in a mold andremoving the more volatile solvent at one temperature and the othersolvent at a higher temperature. For example, the solvents may beremoved by lyophilization.

In general, a synthetic polymer (e.g., p(LGA)) is dissolved in a solventto form a polymer solution having a concentration of between about 5%and about 20% (weight to volume, w/v) (e.g., 5-20%), preferably betweenabout 10% and about 15% (w/v) (e.g., 10-15%), more preferably about12.5% (w/v) (e.g., 12.5%); the molar ratio between the poly(lactic acid)and poly(glycolic acid) in the p(LGA) polymer is between about 20:80 toabout 80:20 (e.g., 20:80-80:20), more preferably about 25:75 (e.g.,25:75). The natural polymer (e.g., pectin) is dissolved in an aqueoussolution to form the second polymer solution having a concentration ofbetween about 0.1% to about 10% (w/v) (e.g., 0.1%-10%), preferablybetween about 0.5% to about 5% (w/v) (e.g., 0.5%-5%), more preferably,about 2% (w/v) (e.g., 2%). The two polymer solutions are then processedas described below to yield a polymeric matrix.

Generally, the matrices are produced by a novel method involving thefollowing: Mixing at least one natural polymer (e.g., pectin) solutionand at least one synthetic polymer (e.g., p(LGA)) solution in acontainer; mixing may be done by vortexing, ultrasonic irradiation,homogenizing, stirring, shaking, rotating or other methods known in theart. Preferably, mixing is accomplished by vortexing at the highestspeed for about 3 to about 5 minutes (e.g., 3-5 minutes). The ratio(volume/volume, v/v) of the two polymer solutions (e.g., pectin/p(LGA))ranges from about 0.1:9.9 to about 9.9:0.1 (e.g., 0.1:9.9 to 9.9:0.1),preferably about 1:9 to about 9:1 (e.g., 1:9 to 9:1), more preferablyabout 1:3 (e.g., 1:3). The resulting emulsified mixture is immediatelypoured into a mold, which was pre-cooled in a bath containing dry iceand isopropyl alcohol, to freeze the mixture quickly.

After freezing, the mold with the contents is transferred into an iceboxfully filled with dry ice and connected to a vacuum line. The vacuumremoves the low molecular weight organic phase leaving the hydrophobicpolymer(s) which are dispersed among the wet-ice phases. This procedureallows a network to form. The temperature in this step is controlled atbetween about −70° C. to about −40° C. (−70° C. to −40° C.), morepreferably about −50° C. (e.g., −50° C.). Then, the temperature of thesystem is raised to between about −20° C. to about −5° C. (e.g., −20° C.to −5° C.), more preferably to about −10° C. (e.g., −10° C.), and thevacuum is continued at about −10° C. (e.g., −10° C.) for an additionaltime to remove the wet-ice phase; this step creates a second polymericnetwork which interpenetrates the first network.

The weight ratio of natural polymer (e.g., pectin) to synthetic polymer(e.g., p(LGA)) is flexible and generally ranges from about 0.2 parts ofnatural polymer (e.g., pectin) to about 99.8 parts of synthetic polymer(e.g., p(LGA) to about 94 parts of natural polymer (e.g., pectin) toabout 6 parts of synthetic polymer (e.g., p(LGA)) (e.g., 0.2 parts ofnatural polymer to about 99.8 parts of synthetic polymer to 94 parts ofnatural polymer to 6 parts of synthetic polymer); preferably about 5parts of natural polymer (e.g., pectin) to about 95 parts of syntheticpolymer (e.g., p(LGA)) (e.g., 5 parts of natural polymer to 95 parts ofsynthetic polymer). Suitable applications for the present matrices(scaffolds) will vary with polymer composition and structure;specifically the matrices (scaffolds) may be used for soft tissue orcartilage or bone repair and regeneration.

The best conditions for pectin/p(LGA) matrix preparation are as follows:concentration of pectin 20 mg/ml; degree of esterification of pectin,90%; concentration of p(LGA), 125 mg/ml; molar ratio between pLA andpGA, 75/25; volume ratio of pectin to p(LGA), 1:3; temperature slots,frozen at −78° C., freeze-dry at −50° C., then at −10° C.

Several parameters enabled an interpenetrating matrix to form by theabove method which does not utilize divalent metal ions (e.g., calciumchloride): (1) control of the concentration of the two polymericsolutions; (2) control of the volume ratio of the two polymericsolutions; (3) mixing (accomplished by vortexing, ultrasonicirradiation, homogenizing, stirring, shaking, rotating or by othermethods); (4) environmental temperature control, minimally the methodrequires two different temperature slots to remove the two solvents byfreeze-drying. Preferably, during removal of the first solvent byfreeze-drying the second solvent should remain frozen. The combinationof these parameters directly determine the degree of interpenetration ofthe two polymers, the thickness of strands in each polymeric networks,the porosity (pore volume, pore size and pore size distribution), andsurface area. In combination these parameters indirectly determine otherproperties of the matrixes, such as mechanical properties, waterstability, enzyme accessibility, mass diffusion and other responses toenvironmental stimuli.

The above method which does not utilize divalent metal ions (e.g.,calcium chloride) can be employed to systems containing three, four ormore types of polymers if these polymers can be dissolved in two or moresolvents which are not miscible, or these polymers have differenttemperature-dependent solubility in the same solvent. Pure water can beadded as the third phase and mixed with the system; the added pure waterfunctions as a pore-forming reagent.

The two types of matrices are different in (1) composition: containingor not containing cross-linkers (e.g. calcium chloride), (2) structure:the natural polymer (e.g., pectin) was ionically cross-linked (e.g., bycalcium for pectin) or the natural polymer is entrapped, (3)application: the first matrix containing cations is not suitable forcartilage generation but is designed for bone regeneration, while thesecond matrix without cations can be used for both bone and cartilagerepair.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described.

The following examples are intended only to further illustrate theinvention and are not intended to limit the scope of the invention asdefined by the claims.

EXAMPLES Example 1

Materials: Sodium salts of pectin from citrus fruits (degree ofesterification, DE, 93%), bovine serum albumin (BSA), fluoresceinamine,p(LGA) [50:50; average M_(w); 50,000-75,000; T_(g) 45-50C], pectinase3XL, neutral-buffered formalin, trypan blue, DNA Quantitation Kit,2,2,2,-trifluoroethanesulfonyl chloride (tresyl chloride), anddimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis,Mo.). Fetal bovine serum, α-minimum essential medium (α-MEM), ascorbicacid-free a-MEM (Formula 94-5049EL), penicillin-streptomycin, Dulbecco'sphosphate-buffered saline, and trypsin-EDTA were purchased from GibcoBRL Products, Life Technologies (Grand Island, N.Y.). Ascorbic acid waspurchased from Fisher Scientific (Pittsburgh, Pa.). Micro BCA® reagentwas from Pierce (Rockford, Ill.). Ethylene oxide was purchased from H.W.Anderson Products (Chapel Hill, N.C.). All other chemicals were A.C.S.grade, and used without further purification.

De-esterification of pectins: Gentle alkaline de-esterification wasperformed by adjusting the pH of a pectin solution (1%, w/v) to 8.0 with0.1N NaOH and stirring at 4° C. over 48 h (Rubinstein, A., et al.,Pharm. Res., 10(2):258-63 (1993)). The reaction solution was dialyzedagainst a large volume of distilled water (DI water). Pectins wererecovered by spreading the pectin solution into ethanol containing 0.1%CaCl₂, the resultant microparticles were filtered, washed with DI water,and lyophilized. Pectin particles with the size ranging from 15 to 125μm were collected. The extent of de-esterification was determined bycomparing the DE values of the de-esterified pectins with those beforethe reaction. The DE values of pectins were measured by high-performanceliquid chromatography (HPLC) (Voragen, A. G. J., et al., FoodHydrocolloids, 1:65-70 (1986)). Other molecular properties of thede-esterified pectins, such as the weight average M_(w); root meansquare radius of gyration (R_(gz)); and intrinsic viscosity ([η]); wereevaluated by HPSEC with on-line multi-angle laser light scattering andviscometric detection (Fishman, M. L., et al., Carbohydrate Res.,5:359-79 (2000)).

Preparation of Pectin/p(LGA) Composites: Pectin/p(LGA) CompositeMatrices were prepared by a multi-step procedure. In step I, 1.0 g ofp(LGA) was dissolved in 8.0 ml of chloroform, into which 0.10 g ofde-esterified pectin, 2.0 g of calcium chloride, and 6.9 g of sodiumchloride were dispersed and blended to form a slurry. The size of theinorganic salt particles ranged from 50 to 200 μm. In step II, theslurry was cast into disks in a mold with dimensions of 6 mm in diameterand 3 mm in thickness, and the solvent was evaporated to form a solidmatrix. In step III, the matrix was immersed in 1 l of deionized water(DI water), where pectin particles started to swell and hydrate, saltsbegan to dissolve and diffuse. Meanwhile, dissolved calcium ions reactedwith and bound to the hydrated pectin particles via inter- andintra-chain chelation. Dissolved sodium chloride and excessive calciumsalts diffused to create spaces for water migration. The process in stepIII was continued for 48 h. In that time the water was changed severaltimes to complete cross-linking of pectin and leaching of residualsalts. Lastly, freeze drying the matrices created a channeled porousstructure.

Porous p(LGA) matrices were prepared by the same method as describedabove, except for the substitution of pectin with sodium chloride.Porous pectin matrices were prepared by casting pectin solution (2.0%,w/v) in a mold (6×3 mm (d×h)) lyophilizing the solution to create asolid structure, which thereafter was treated with calcium chloridesolution (0.1 m) and lyophilized. The p(LGA) and pectin matrices wereused as controls.

Recovery of p(LGA) and pectin from pectin/p(LGA) matrices: Samples wereanalyzed to determine the efficiency with which calcium chloridecross-linked pectin particles and the amounts of pectin and p(LGA) inthe final composite matrices. Samples of pectin/p(LGA) matrices werevacuum-dried for 24 h prior to experimentation. Each dried sample wasplaced in 2.0 ml tetrahydrofuran (THF) in a volumetric flask equippedwith a penny head stopper to prevent solvent evaporation. The mixturewas continually shaken at low speed for 2 h to complete the extractionof p(LGA) polymers. The extraction solution was removed and analyzed forp(LGA) content using a Shimadzu HPLC equipped with an RID-10A refractiveindex detector and an SCL-10A data station (Model LC-10AD, Kyoto,Japan). An aliquot of the solution (10 ml) was injected and eluted byTHF on a phenogel guard column (model 22824G, 50×7.8 mm, Phenomenex,Torrance, Calif.) and a phenogel column (model GP/4446, 300×7.8 mm,Phenomenex) at the flow rate of 0.5 ml/min. p(LGA)/THF solutions ofknown concentrations were run under the same conditions and used toprepare a standard curve.

After the removal of p(LGA) polymers, the solid residues, calciumcross-linked pectins, were washed with fresh THF (2×2 ml), dry ethanol(3×2 ml), and air-dried. Sodium phosphate solution (1.0 M, 2.0 ml, pH6.5) was added to the flask which was sonicated to solubilize thepectin. Pectin content was analyzed by total sugar assay (Dubois, M., etal., Anal. Chem., 28:350-6 (1956); Liu, L. S., and R. A. Berg, J.Biomed. Mater. Res. (Appl. Biomater.), 63:326-32 (2002)).

Chemical modification of pectin/p(LGA) matrices: The chemicalmodification of pectin/p(LGA) composite matrices was performed bygrafting the matrices with fluoresceinamine using tresyl chloride as acoupling reagent (Nilsson, K., and K. Mosbach, Biochem. Biophys. Res.Comm., 102(1):449-57 (1981); Dickerson, K. T., et al., U.S. Pat. No.5,677,276). Samples of pectin/p(LGA) matrix were immersed in dry acetone(pre-dried over molecular sieve 4A; Acros, Pittsburgh, Pa.) for 24 hwith three changes. To a glass vial containing 2.0 ml dry acetone andone piece of the dry sample, pyridine (200 ml) and tresyl chloride (100ml) were added, and gently shaken for 10 min at room temperature. Thesample was removed and rinsed with dry acetone (3×5 ml),phosphate-buffered saline (PBS) (pH 7.0, 2×5 ml), and placed in 2.0 mlof PBS (pH 7.8) containing fluoresceinamine (20 mm) and incubated for 20h at room temperature. To completely remove the fluoreceinamine whichwas physically adsorbed rather than chemically conjugated, the samplewas washed with 1 mM HCl and 0.2 M NaHCO₃ repeatedly, and 1.0 M NaClcontaining 1 mM HCl, finally with DI water (U.S. Pat. No. 5,677,276).Samples thus treated were examined by confocal laser fluorescencemicroscopy as described in the following section.

P(LGA) matrices treated with both tresyl chloride and fluoreceinamineunder the same conditions were used as controls.

Microscopic imaging. Scanning electron microscopy (SEM): For SEMexaminations, samples of pectin particles, NaCl—CaCl₂ crystal mixtures,pectin/p(LGA), and p(LGA) matrices were mounted on specimen stubs,coated with a thin layer of gold in a sputter coating apparatus (EdwardsHigh Vacuum, Wilmington, Mass.), and examined in a model JSM 840Ascanning electron microscope (Jeol USA, Peabody, Mass.) operating at 10kV in the secondary electron imaging mode. Images were collected at 25×and 250× using an Imix-1 digital image workstation (PrincetonGamma-tech, Princeton, N.J.). Confocal laser microscopy: Samples offluorescently labeled pectin/p(LGA) composite matrices were glued to 1×3inch microscope slides and placed in the sample stage of an IRBE opticalmicroscope with a 10× lens integrated with a model TCS-SP laser scanningconfocal microscope (Leica Microsystems, Exton, Pa.). The parameters forthe image acquisition were set for confocal reflection (633 mm) andconfocal fluorescence (488/500-530 nm) in two channels.

Dynamic mechanical analysis (DMA): Compressive mechanical testing of thematrices was performed on a Rheometric Scientific RSA II Solids Analyzer(Rheometric Scientific, Piscataway, N.J.) fitted with 25 mm parallelplates. Temperature control was maintained using a liquid nitrogenenvironmental controller. Each sample matrix was placed on the lowerplate, the upper plate was lowered onto the sample to give a slightcompressive force, and then locked in the place. The samples were testedusing a compressive strain of 0.25-1.0%, depending on the stiffness ofthe sample. Storage modulus, loss modulus, and loss tangent weredetermined over a temperature range of −100° C. to +200° C. at theheating rate of 10° C./min. The data were analyzed using RheometricScientific Orchestrator software, version 6.5.7.

Determination of equilibrium water content and protein adsorption:Samples of pectin/p(LGA) and p(LGA) matrices were dried under vacuum atroom temperature for 24 h. prior to the experiment. Each dried samplewas incubated with a large volume of PBS (pH 7.0) at room temperatureunder gentle shaking. Samples were removed from the incubation solutionsat intervals of 5, 15, 30, 45 min, and 1, 2, 4, 8 h., rinsed three timeswith DI water, wiped with tissue paper to remove the water adsorbed onthe surfaces, and weighed (W_(w)): The samples were then re-dried undervacuum for 24 h. and weighed (W_(rd)): The water content of matrices ateach time point was calculated: water content=(W_(w)×W_(rd))=W_(w)×100%.

The kinetics of protein adsorption in pectin/p(LGA) and p(LGA) matriceswas studied by a procedure similar to that used for the determination ofequilibrium water content, except for the addition of BSA (0.1%, w/v) inPBS. After rinsing with DI water, the samples were analyzed for theamount of protein adsorbed by protein BCA assay (Smith, P. K., Anal.Biochem., 150:76-85 (1985)). A series of BSA solutions with knownconcentrations were used to prepare a standard curve.

In vitro cell culture and bioassays: The potential for application ofcomposite matrices in tissue engineering was evaluated in vitro byseeding and culturing osteoblast cells on the matrices. Osteoblasts(MC3T3-E1, clone 26) were thawed, cultured in a supplemented ascorbicacid-free α-MEM and 10% fetal calf serum (FBS) containing 100 U/mlpenicillin and 100 mg/ml streptomycin in a humidified incubator at 37°C. with 5% CO₂. The medium was changed every other day. The cells ofpassages 3 and 4 were harvested, pelleted by centrifugation andre-suspended at the concentration of 2×10⁶ cell/ml in α-MEM containingFBS (10%), antibiotics (1%), and L-ascorbic acid (50 mg/l) (completemedium). The viability of the cells was higher than 90% as determinedwith the trypan blue exclusion assay.

The pectin/p(LGA) and p(LGA) matrices were sliced into disks withdimensions of 6 mm in diameter and 1.5 mm in thickness, and sterilizedin culture flasks with ethylene oxide for 2 days. The matrices weresoaked in ethanol for 30 min, exchanged with PBS for three times for 30min each time, then washed with the complete medium twice for 2 h eachtime.

For the cell attachment test, each of the matrices were placed in ateflon plate containing 0.5 ml of the cell suspension, cultured on anorbital shaker (Model 3520; Lab-Line Instrument, Melrose Park, Ill.) at75 rpm under standard conditions. At day 3, the cell-loaded matriceswere transferred into six-well tissue culture plates, 4 ml of completemedium were added into each well, cultured under standard conditions for1 day. The matrices were removed from the medium, washed with PBS, fixedin 10% neutral-buffered formalin, dehydrated, and embedded in paraffinusing standard procedures. Paraffin-embedded specimens were sectionedinto 5-μm thick through the center, stained with hematoxylin and eosin,and examined under a light microscope (Lanza, R. P., et al., Principlesof tissue engineering, Academic Press, San Diego, Calif., 1997; Carson,F. L., Histotechnology: a self instructional text, ASCP Press, Chicago,Ill., 1990).

For cell proliferation studies, the cell culture was continued for anadditional 7 and 14 days. The medium was changed every other day. At theconclusion of cell culture, the matrices were removed, washed with PBS,homogenized using a polytron homogenizer (Brinkmann Easycare Generator;Polytron-Aggregate, Switzerland) for 30 s at top speed (VI) for threetimes, then subjected to DNA assay for cell number quantitation (Lanza,R. P., et al., Principles of tissue engineering, Academic Press, SanDiego, Calif., 1997; Patrick, C. W., et al., editors, Frontiers intissue engineering, Pergamon, N.Y., 1998). DNA assays were performedusing DNA Quantitation Kit with Hoechst 33258 dye. The concentration ofDNA in solution was converted to a cell number using a conversion factorof 7.8 pg of DNA per MC3T3-E1 cell. This conversion factor wasdetermined by measuring the amount of DNA from a known cell number.

Statistical analysis: The data presented here are mean ±standarddeviation. To test the significance of observed differences between thestudy groups, a paired Student's t-test was applied.

Results and discussion. De-esterification of pectin: Pectins werede-esterified prior to use for preparation of pectin/p(LGA) compositematrices. An almost complete de-esterification was accomplished(Table 1) to enable the insolubilization of pectin macromolecules bycalcium ions. The M_(w) and intrinsic viscosity of the pectin wereslightly reduced after de-esterification in comparison with the originalpolymer (Table 1). In addition, R_(gz) of pectin was reduced slightlyafter its de-esterification (Table 1). This indicated that somedisaggregation and/or degradation of pectin macromolecules occurredduring its de-esterification. Nevertheless, this seems to notsignificantly effect the binding efficiency of pectin to calcium ionssince more than 80% of the pectin suspended in the p(LGA)/chloroformsolution was recovered from the resulting pectin/p(LGA) matrices. Alsothe ratio of pectin to p(LGA) polymers was reduced only slightly aftermatrix fabrication (Table 2).

De-esterified pectins in the form of microparticles were collected andused for pectin/p(LGA) matrix preparation. The pectin particles showedsome variations in size and morphology (FIG. 1A).

Preparation and microscopy of pectin/p(LGA) matrices: In the processdescribed herein to prepare pectin/p(LGA) composite matrices, in onestep the salt particles function passively as pore-creating reagents andsimultaneously the salts play the additional role of a cross-linker, tobridge the highly de-esterified pectin particles. When a dry, solidmatrix of polymer/salt was immersed in water, sodium chloride andcalcium chloride dissolved quickly and diffused toward the surroundingliquid phase. Simultaneously, pectin molecules hydrated and swelledslowly as determined by its viscoelastic nature. Normally, the swelledpectin molecules would tend to dissolve and diffuse into the water phasebut they were stopped by the cross-linking with calcium ions to formpectin-calcium hydrogels. The insolubilization of pectin was confirmedby analyzing it in the final matrix (Table 2).

The organization and microstructure of p(LGA) and pectin/p(LGA) matricesare illustrated in FIG. 2. Both matrices were porous and had asponge-like morphology. Pores were evenly distributed into all areas ofthe matrices, and they were interconnected. The pores in p(LGA) matriceshad sizes and shapes that matched those of the original salt crystals,as evidenced by SEM (FIGS. 1B and 2A). Pores and channels in p(LGA)matrices were lined by fibrils or flakes of p(LGA) polymers, indicatingthe deposition of p(LGA) polymers in the gaps or crevices among saltparticles as the solvent evaporated. In this case, the microstructureand morphology of the matrices only depends on the weight ratio ofsalts/polymers and on the particle size of the salt.

Topographical, SEM images also revealed that the pores of the compositepectin/p(LGA) matrices were often smaller than the p(LGA) alone, andthey were always lined or bordered by smooth, leaf-like surfaces (FIG.2B). These surfaces resembled the appearance of the image texture ofisolated pectin particles used to make the composite (FIG. 1A). Withoutbeing bound by theory, these images suggest that the p(LGA) forms aparenchymal matrix, binding the pectin particles which line the pores,together in the composite; nevertheless, it is difficult to localize thep(LGA) based on image features alone.

Pectin particles were covalently tagged with fluoresceinamine in orderto locate areas of p(LGA) indirectly in the composite matrix. Confocalfluorescence and confocal reflection microscopy of the surfaces ofcomposite matrices were used to resolve microscopically the integratedorganization of the two components in correlated images. Reflectionimages (FIG. 3A) in stereo projection reveal the composite structure. Afew flat areas of reflection coincided with areas of green fluorescenceor pectin (FIG. 3B) which indicated that whether or not areas of pectinparticles reflected light depended upon their orientation. Other areasof reflection, containing irregular tubes and anastomoses do notfluoresce, indicating that these areas contain p(LGA) (FIG. 3A).

Without being bound by theory, from the above observations it appearsthat the pectin domains not only filled in the pore spaces created bythe deposition of p(LGA) polymers in gaps among particles, but alsocovered or wrapped most of the p(LGA) domains. In general, pectin/p(LGA)matrices presented a complex structure of connected porous pectinnetworks which were reinforced by p(LGA) networks.

Dynamic mechanical properties of pectin/p(LGA) matrices: Dynamicmechanical analysis (DMA) is a useful complement to microscopic methodsfor morphology and microstructure investigations of polymericcomposites. The dynamic mechanical properties of the pectin/p(LGA)composite matrix and p(LGA) matrix were determined by measuring theircompressive storage modulus (E′), loss modulus (E″), and loss tangent(tan d). Typical compressive curves are compared for each sample in FIG.4, along with curves for the pectin alone. The p(LGA) matrix exhibited anoticeable drop in storage modulus starting at about −80° C. which thenplateau by about −40° C. It also had a sharp glass transition at about50° C., which is consistent with the data obtained from the supplier.Above this temperature the sample no longer gave any force readings onthe instrument. A sharp peak at about −80° C. was seen in the lossmodulus curve and the loss tangent curve. The overall trends for thepectin/p(LGA) composite curves are similar to those for p(LGA) curves.Nevertheless, significant differences were noted. The pectin/p(LGA)composite showed a much smaller decline in storage modulus over the −80°C. to −40° C. range, and had a higher value for the storage modulus overthe entire temperature range. It too showed the glass transition atabout 50° C. However, above this temperature the sample still maintainedseveral grams of residual force, whereas the p(LGA) alone retainedvirtually none. The −80° C. peak in the loss modulus was also stillvisible in the composite, although it was much smaller than in thep(LGA) matrix, and seemed to be smaller than what would be expected fromcompositional differences alone.

The curves for plain pectin matrices were relatively flat and werecomparable to DMA curves obtained for neat pectin films undergoing smalldeformation dynamic stretching motion [24]. Pectin undergoing dynamicsmall deformation compression had lower values of storage modulus andloss modulus than the other two matrices below 50° C. However, at about50° C. the curves for pectin/p(LGA) and p(LGA) matrices had dropped tobelow the value of the pectin matrix.

The loss tangent behavior of pectin/p(LGA) and p(LGA) matrices wassimilar to that seen with the loss modulus, although the difference inthe peak size at −80° C. was more striking. The pectin curve showed abroad peak at about 30° C.

Based on these data, it appears that the presence of the pectin raisesthe mechanical stiffness of the matrix above that of the p(LGA) matrixby itself at temperatures below the glass transition. Above the glasstransition, the pectin seems to enable the matrix to maintain some levelof physical integrity, although this is at a much lower level than forthe matrix at temperature below the glass transition. The incorporationof the pectin network structure seems to be primarily responsible forthe increase in the values for E0 and for the decrease in the tan valuesfor the pectin/p(LGA) composite matrix compared to the p(LGA) matrix.These differences are considered to be the contribution of thewell-organized double-network structure of the composite matrices, wherethe thermoplastic p(LGA) networks were reinforced by thenon-thermoplastic pectin networks. The presence of the pectin in thematrix was instrumental in limiting molecular motion of p(LGA) polymerswith increasing temperature.

Characterization of pectin/p(LGA) matrices as carriers of signalmolecules: Pectin/p(LGA) matrices were evaluated as carriers of signalmolecules by conjugating the matrix with fluoresceinamine (FIG. 3).Green fluorescence was localized in irregular sheets and patches (FIG.3B). These fluorescent structures were similar to those revealed by SEMfor pectin/p(LGA) composite matrices (FIG. 2B), indicating the graft ofthe fluoresceinamine in pectin areas. Fluorescence was absent from thep(LGA) areas of fibrillar networks in the composite matrices (cf. FIGS.3A and B). This was consistent with the lack of fluorescent emissionobserved for p(LGA) matrices (data not shown), indicating the inertnature of p(LGA) to the immobilization reaction. The signal molecules offluoresceinamine were conjugated directly to the sugar rings of pectinvia the activation of the hydroxyl groups of the pectins. The hydroxylgroups of carbohydrate molecules are only mildly nucleophilic,approximately equal to water in their relative nucleophilicity. Thus,the activation of pectins was performed in dry acetone to formintermediate reactive derivatives containing good leaving groups fornucleophilic substitution. The reaction of activated hydroxyls withnucleophiles was conducted in PBS (pH 7.8) at room temperature, whichresulted in stable covalent bonds between the carbohydrate and theamine-containing molecules. Tresyl chloride has been demonstrated to bea useful tool to conjugate various peptides and proteins with syntheticpolymers or natural polymers. Nevertheless, we observed some loss inmatrix integrity in the current experiment, especially when the matriceswere treated with dry acetone and during the repeated washing process;without being bound by theory, it may be due to the differences inswellability between the two networks with medium changes.

Since most signal molecules are environmentally sensitive, theincorporation of signal molecules into biomedical devices is often doneunder very mild conditions such as in aqueous media, at neutral solutionpH, and at 37° C. or lower. We evaluated the potential for compositematrices to adsorb signal molecules from aqueous solution by measuringthe equilibrium water content and the amount of adsorbed protein. Thetotal water content of matrices was determined by swelling samples ofeach matrix in PBS and measuring the increase in weight at eachincubation time point (FIG. 5A). There was an increase in the watercontent with the incubation time for both types of matrices. Due to theinclusion of a hydrophilic network, pectin/p(LGA) matrices facilitatedwater diffusion and uptake into the matrices, as demonstrated by a quickincrease in matrix weight at the beginning of incubation. Less time isrequired to reach equilibrium, and a higher percentage of water adsorbedover the entire time of incubation in comparison with p(LGA) matrices.At steady state, the water content of pectin/p(LGA) composite matriceswas about eight-fold of that of p(LGA) matrices (FIG. 6). For proteinadsorption, there was a trend similar to water uptake for bothpectin/p(LGA) and p(LGA) matrices (FIG. 5B). As in the case of water,the pectin/p(LGA) matrix adsorbed more protein than the p(LGA) matrix(FIG. 6). However, the adsorbed BSA found in pectin/p(LGA) matrices wasonly 1.5-fold of that detected in p(LGA) matrix (FIG. 6). For p(LGA)matrices, both water and BSA are only able to diffuse to and remain inpore spaces of the matrices. In pectin/p(LGA) matrices, small moleculesof water not only diffused and remained in the pore spaces, but alsopenetrated into the pectin gel domains. Compared to water penetration,only a small fraction of protein BSA was capable of penetrating to thepectin domains. These results demonstrated the capability ofpectin/p(LGA) composite matrices to carry signal molecules either bychemical conjugation or by physical adsorption.

In vitro cell culture: After 1 day of cell seeding, osteoblasts wereattached onto pectin/p(LGA) and p(LGA) matrices. There were more cellson pectin/p(LGA) matrices than on p(LGA) matrices. Furthermore,histological analysis revealed that osteoblasts attached topectin/p(LGA) matrices in multi-layers whereas they attached to p(LGA)matrices in a single layer (FIG. 7). Cells were not only attached onthese matrices, but were also viable and had the capability toproliferate (FIG. 8). Although the difference in cell number was notstatistically significant at the beginning, after 2 weeks culture, cellnumbers on the pectin/p(LGA) matrices were two-fold of that on thep(LGA) matrices (FIG. 8).

Conclusions: We have presented a method to effectively combine syntheticpolymers (e.g., p(LGA)) and natural polymers (e.g., pectin) in onematrix. By including dry particles of pectins and calcium chloride inp(LGA)/chloroform solution, composite matrices were created with aninterconnected porous morphology. The composite matrices consisted of apectin network reinforced by a p(LGA) network. The composite matricescombined the best features of both polymers. Typically, the mechanicalproperties of the composite were comparable to p(LGA) whereas theircapacity to hold water and accessibility to proteins were comparable topectin. In addition, the composite matrices provided side chainfunctional groups for further chemical modifications, which could beused in various biomedical applications. As demonstrated by in vitrocell culture, the composite matrices show promise for tissue engineeringapplications. Thus, by selecting a group of synthetic polymers withappropriate pairs of inorganic salts and polysaccharides, many polymericcomposite matrices can be created by this simple and environmentallyfriendly method.

Thus, in one aspect, the method described herein for producing thematrices is a one step process for creating pores and chemicalcross-links in a pectin-based material by pre-loading CaCl₂ salts intothe precursor suspension of pectin/p(LGA). The resulting pectin/p(LGA)composites possess a series of unique features different from matricesof p(LGA) alone. Namely, these composites allow hydrophilic substancesto access and affiliate to them. This feature is important in preparingcarriers for protein delivery: DNA therapeutics, implants for tissuerepair, and other health care products. In these applications, devicesprepared from p(LGA) alone lacked efficacy due to their hydrophobicnature. It is expected that pectin/p(LGA) composites will be usefuldelivery vehicles for a larger number of protein drugs, which areorganic solvent sensitive.

Example 2

Matrix preparation using pectin and poly(lactide-co-glycolide)(p(LGA)).Pectin was dissolved in D.I. water at the concentration of 1-50 mg/ml;p(LGA) was dissolved in dichloromethane at 10-200 mg/ml; the twosolutions were mixed at the volume ratio of 1/10 or 10/1 (pectin:p(LGA)) by vortexing. The mixture was poured into a mold, which waspre-cooled at the temperature lower than −75° C. (using liquid nitrogenor the mixture of dry ice and isopropyl alcohol). After the slurry wasfrozen, the mold was transferred into a desiccator which was placed inan icebox containing dry ice (−56.6° C.) and connected to a vacuum line.The icebox was evacuated for 8 hrs at −56.6° C., followed by 12 hrs inlyophilizer at −10° C.

All of the references cited herein are incorporated by reference intheir entirety. Also incorporated by reference in their entirety are thefollowing references: BeMiller, J. M., An introduction to pectins:structure and properties, In: Chemistry and function of pectins, ACSseries 310, M. L. Fishman and J. J. Jen, editors, American ChemicalSociety, Washington, D.C., 1986, p. 2-13; Berhold, et al., J. Cont.rRel., 39:17-25 (1996); Bodmeier, R., and O. Paeratakul, Pharm. Res.,11:882-888 (1994); Carson, F. L., Histotechnology: a self instructionaltext, ASCP Press, Chicago, Ill., 1990; Chen, et al., Science,276:1425-1428 (1997); Coffin, D. R., et al., J. Appl. Polym. Sci.,61:71-79 (1996); Dickerson, K. T., et al., U.S. Pat. No. 5,677,276;Dubois, M., et al., Anal. Chem., 28: 350-356 (1956); Fishman, M. L., etal., Carbohydrate Res., 5:359-379 (2000); Fishman, M. L., et al., J.Agr. Food Chem., 49: 4494-4501 (2001); Freed, et al., J. Biomed. Mater.Res., 27:11-23 (1993); Hwang, J., and J. L. Kokini, J. Texture Stud.,22: 123-167 (1991); Ishaug et al., J. Biomed. Mater. Res., 36:17-28(1997); Kim, J. H., and R. Fassihi, J. Pharm. Sci., 86(3): 316-328(1997); Langer, R., J. P. Vacanti, Science, 260: 920-926 (1993); Lanza,R. P., et al., Principles of tissue engineering, Academic Press, SanDiego, Calif., 1997; Liu, L. S., and R. A. Berg, J. Biomed. Mater. Res.(Appl. Biomater.), 63: 326-332 (2002); Liu, L. S., et al., Biomaterials,24: 3333-3343 (2003); Liu, L. S., et al., Conversion of Pectin andRelated Polysaccharides into Unique Biomaterials for BiomedicalApplications, Proceedings of the United States-Japan UJNR CooperativeProgram in Natural Resources and Agriculture Panel, 32^(nd) AnnualMeeting, Tsukuba, Ibaraki, Japan, Nov. 9-15, 2003, pages 405-409; Liu,L. S., et al., Biomaterials, 25: 3201-3210 (2004); Ma, P. X., and R.Langer, Fabrication of biodegradable polymer foams for celltransplantation and tissue engineering, In: Tissue engineering methodsand protocols, J. Morgan and M. Yarmush, editors, Humana Press Inc.,Totowa, N.J., 1999, p. 47-56; Ma, P. X., et al., J. Biomed. Mater. Res.,54(2): 284-293 (2001); Massia, S. P., and J. A. Hubbell, Anal. Biochem.,187: 292-301 (1990); Mikos, et al., J. Biomed. Mater. Res., 27:183-189(1993); Mikos, A. G., et al., Polymer, 35(5): 1068-1077 (1994); Nilsson,K., and K. Mosbach, Biochem. Biophys. Res. Comm., 102(1): 449-457(1981); Ouchi, T. et al, Macromolecules, 3(5): 885-888 (2002); Patrick,C. W., et al., editors, Frontiers in tissue engineering, Pergamon, N.Y.,1998; Rubinstein, A., et al., Pharm. Res., 10(2): 258-263 (1993);Schols, H. A., and A. G. J. Voragen, Complex pectins: structureelucidation using enzymes, In: Pectin and pectinases, J. Visser and A.G. J. Vorangen, editors, Elsevier Science, Amsterdam, 1996, p. 3-19;Semde, R., et al., Int. J. Pharm., 197: 181-92 (2000); Smith, P. K.,Anal. Biochem., 150: 76-85 (1985); Temenoff et al., Biomaterials,21:431-440 (2000); and Voragen, A. G. J., et al., Food Hydrocolloids, 1:65-70 (1986).

Also incorporated by reference in their entirety are the following U.S.Pat. Nos. 4,060,081; 5,041,138; 5,514,378; 5,744,516; 5,817,728;6,010,870; 6,114,496; 6,124,384; 6,150,438; 6,207,749; 6,294,202;6,326,021; 6,350,531; 6,379,962; 6,388,047; 6,399,700; and 6,423,345.

Thus, in view of the above, the present invention concerns (in part) thefollowing:

A porous polymeric matrix (polymeric composition) comprising (orconsisting essentially of or consisting of) at least one natural polymerand at least one synthetic polymer and optionally at least one cation.

The above porous polymeric matrix, wherein said synthetic polymer isselected from the group consisting of polyesters, polyanhydrides,polyortho esters, and mixtures thereof.

The above porous polymeric matrix, wherein said polyester is selectedfrom the group consisting of poly(lactic acid), poly(glycolic acid),poly(lactic acid-co-glycolic acid), poly(lactide), poly(glycolide),poly(lactide-co-glycolide), polycaprolactone,poly(lactide-co-caprolactone), and mixtures thereof.

The above porous polymeric matrix, wherein said polyester ispoly(lactide-co-glycolide) or polycaprolactone.

The above porous polymeric matrix, wherein saidpoly(lactide-co-glycolide) has a MW from about 500 to about 10,000,000Da.

The above porous polymeric matrix, wherein saidpoly(lactide-co-glycolide) has a MW from about 2,000 to about 1,000,000Da.

The above porous polymeric matrix, wherein saidpoly(lactide-co-glycolide) has a MW from about 500 to about 5,000 Da.

The above porous polymeric matrix, wherein saidpoly(lactide-co-glycolide) has a LA:GA ratio of from about 75:25 toabout 85:15 (mol:mol).

The above porous polymeric matrix; wherein said polyanhydride isselected from the group consisting of poly(carboxyphenoxypropane-sebacic acid), poly(1,6-bis(p-carboxyphenoxy)hexane,poly(anhydride-co-imide), and mixtures thereof.

The above porous polymeric matrix, wherein said polyortho ester isselected from the group consisting of ALZAMER, CHRONOMER, copolymers ofALZAMER or CHRONOMER with poly(lactide-co-glycolide) or polyethyleneglycol, and mixtures thereof.

The above porous polymeric matrix, wherein said natural polymer ispectin.

The above porous polymeric matrix, wherein said pectin has a MW fromabout 500 to about 1,000,000 Da.

The above porous polymeric matrix, wherein said pectin has a MW fromabout 230,000 to about 280,000 Da.

The above porous polymeric matrix, wherein said pectin has a MW of about3000 Da.

The above porous polymeric matrix, wherein said pectin has a DE fromabout 10 to about 100%.

The above porous polymeric matrix, wherein said pectin has a DE fromabout 25 to about 76%.

The above porous polymeric matrix, wherein said pectin has a DE fromabout 25 to about 35%.

The above porous polymeric matrix, wherein said porous polymeric matrixdoes not contain a cation.

The above porous polymeric matrix, wherein said porous polymeric matrixcontains at least one cation.

The above porous polymeric matrix, wherein said cation is selected fromthe group consisting of calcium, sodium, magnesium, ammonium, andmixtures thereof.

The above porous polymeric matrix, wherein said cation is selected fromthe group consisting of calcium, sodium, and mixtures thereof.

A method of making a porous polymeric matrix, comprising (or consistingessentially of or consisting of) mixing at least one natural polymer andinorganic salts with a solution comprising (or consisting essentially ofor consisting of) at least one solvent and at least one syntheticpolymer to form a slurry, casting said slurry in a mold and removingsaid solvent to form solid matrices, immersing said solid matrices indeionized water to allow natural polymer cross-linking and pore creationto occur simultaneously, and drying said matrices to create a porouspolymeric matrix; wherein said porous polymeric matrix comprises atleast one natural polymer and at least one synthetic polymer and atleast one cation.

The above method, wherein the ratio of said salt particles and saidnatural polymer to said synthetic polymer is about 1:1 to about 40:1.

The above method, wherein said inorganic salts are selected from thegroup consisting of calcium chloride, sodium chloride, magnesiumchloride, sodium sulfate, potassium sulfate, ammonium sulfate, ammoniumchloride, potassium chloride, and mixtures thereof.

The above method, wherein the ratio of said natural polymer to saidinorganic salts is from about 1:about 0.1-about 20.

The above method, wherein the ratio of said natural polymer to saidsynthetic polymer is from about 0.1:about 99.9 to about 99.9: about 0.1.

The above porous polymeric matrix, wherein said porous polymeric matrixis made by the above method.

The above porous polymeric matrix, wherein said porous polymeric matrixis made by the method below.

A porous polymeric matrix comprising (or consisting essentially of orconsisting of) at least one natural polymer and at least one syntheticpolymer and at least one cation, wherein said porous polymeric matrix ismade by a method comprising (or consisting essentially of or consistingof) mixing at least one natural polymer and inorganic salts with asolution comprising at least one solvent and at least one syntheticpolymer to form a slurry, casting said slurry in a mold and removingsaid solvent to form solid matrices, immersing said solid matrices indeionized water to allow natural polymer cross-linking and pore creationto occur simultaneously, and drying said matrices to create a porouspolymeric matrix.

A method of making a porous polymeric matrix, comprising (or consistingessentially of or consisting of) mixing at least one natural polymer inan aqueous solvent and mixing at least one synthetic polymer in anorganic solvent, combining the mixtures and casting in a mold, andseparately removing said aqueous solvent and said organic solvent toform a porous polymeric matrix; wherein said porous polymeric matrixcomprises at least one natural polymer and at least one syntheticpolymer and does not contain a cation.

A porous polymeric matrix comprising (or consisting essentially of orconsisting of) at least one natural polymer and at least one syntheticpolymer, wherein said porous polymeric matrix is made by a methodcomprising (or consisting essentially of or consisting of) mixing atleast one natural polymer in an aqueous solvent and mixing at least onesynthetic polymer in an organic solvent, combining the mixtures andcasting in a mold, and separately removing said aqueous solvent and saidorganic solvent to form a porous polymeric matrix; wherein said porouspolymeric matrix does not contain a cation.

A method for making an osteoblast containing porous polymeric matrix,comprising (or consisting essentially of or consisting of) providing theabove porous polymeric matrix in a nutrient environment and attachingosteoblast cells to said porous polymeric matrix to form an osteoblastcontaining porous polymeric matrix suitable for implantation into apatient to replace defective or missing bone.

A method for engineering tissue comprising (or consisting essentially ofor consisting of) growing cells on the above porous polymeric matrix.

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of this specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

TABLE 1 Molecular properties of pectins De-esterification PropertiesBefore After Mw × 10⁻⁵ 0.87 (0.02) 0.81 (0.01) Rgz (nm) 24.7 (2.06) 21.4(0.06) [η]_(w) (dL/g) 1.25 (0.02) 1.22 (0.02) DE (%) 93 10.2 (0.77)

TABLE 2 Physical characterization of pectin/p(LGA) and p(LGA) matricesDensity Pectin content (mg) P(LGA) content (mg) Matrices (g/ml)calculated determined calculated determined Pectin/ 0.190 1.28 1.06 ±0.2 12.8 11.4 ± 3.2 p(LGA) P(LGA) 0.306 N/A N/A 26.0 24.8 ± 2.4

1. A method of making a porous polymeric matrix, comprising mixing atleast one natural polymer and inorganic salts with a solution comprisingat least one solvent and at least one synthetic polymer to form aslurry, casting said slurry in a mold and removing said solvent to formsolid matrices, immersing said solid matrices in deionized water toallow natural polymer cross-linking and pore creation to occursimultaneously, and drying said matrices to create a porous polymericmatrix; wherein said porous polymeric matrix comprises at least onenatural polymer and at least one synthetic polymer and at least onecation.
 2. The method according to claim 1, wherein the ratio of saidsalt particles and said natural polymer to said synthetic polymer isabout 1:1 to about 40:1.
 3. The method according to claim 1, whereinsaid inorganic salts are selected from the group consisting of calciumchloride, sodium chloride, magnesium chloride, sodium sulfate, potassiumsulfate, ammonium sulfate, ammonium chloride, potassium chloride, andmixtures thereof.
 4. The method according to claim 1, wherein the ratioof said natural polymer to said inorganic salts is from about 1:about0.1-about
 20. 5. The method according to claim 1, wherein the ratio ofsaid natural polymer to said synthetic polymer is from about 0.1:about99.9 to about 99.9:about 0.1.
 6. A porous polymeric matrix comprising atleast one natural polymer and at least one synthetic polymer and atleast one cation, wherein said porous polymeric matrix is made by amethod comprising mixing at least one natural polymer and inorganicsalts with a solution comprising at least one solvent and at least onesynthetic polymer to form a slurry, casting said slurry in a mold andremoving said solvent to form solid matrices, immersing said solidmatrices in deionized water to allow natural polymer cross-linking andpore creation to occur simultaneously, and drying said matrices tocreate a porous polymeric matrix.
 7. The porous polymeric matrixaccording to claim 6, wherein said cation is selected from the groupconsisting of calcium, sodium, magnesium, ammonium, and mixturesthereof.
 8. The porous polymeric matrix according to claim 6, whereinsaid cation is selected from the group consisting of calcium, sodium,and mixtures thereof.
 9. The porous polymeric matrix according to claim1, wherein said synthetic polymer is selected from the group consistingof polyester, polyanhydride, polyortho ester, and mixtures thereof. 10.The porous polymeric matrix according to claim 9, wherein said polyesteris selected from the group consisting of poly(lactic acid),poly(glycolic acid), poly(lactic acid-co-glycolic acid), poly(lactide),poly(glycolide), poly(lactide-co-glycolide), polycaprolactone,poly(lactide-co-caprolactone), and mixtures thereof.
 11. The porouspolymeric matrix according to claim 10, wherein said polyester ispoly(lactide-co-glycolide).
 12. The porous polymeric matrix according toclaim 11, wherein said poly(lactide-co-glycolide) has a MW from about500 to about 10,000,000 Da.
 13. The porous polymeric matrix according toclaim 11, wherein said poly(lactide-co-glycolide) has a MW from about2,000 to about 1,000,000 Da.
 14. The porous polymeric matrix accordingto claim 11, wherein said poly(lactide-co-glycolide) has a MW from about500 to about 5,000 Da.
 15. The porous polymeric matrix according toclaim 11, wherein said poly(lactide-co-glycolide) has a LA:GA ratio offrom about 75:25 to about 85:15 (mol:mol).
 16. The porous polymericmatrix according to claim 9, wherein said polyanhydride is selected fromthe group consisting of poly(carboxyphenoxy propane-sebacic acid),poly(1,6-bis(p-carboxyphenoxy)hexane, poly(anhydride-co-imide), andmixtures thereof.
 17. The porous polymeric matrix according to claim 9,wherein said polyortho ester is selected from the group consisting ofALZAMER, CHRONOMER, copolymers of ALZAMER or CHRONOMER withpoly(lactide-co-glycolide) or polyethylene glycol, and mixtures thereof.18. The porous polymeric matrix according to claim 10, wherein saidpolyester is polycaprolactone.